Tumor-associated macrophages can release a vast diversity of growth factors, proteolytic enzymes, cytokines, and inflammatory mediators. Many of these factors are key agents in cancer metastasis. Daintain/AIF-1 is a macrophage-derived inflammatory cytokine which defined a distinct subset of tumor-associated activated macrophages/microglial cells. Previous study demonstrated that daintain/AIF-1 could promote breast cancer proliferation through activating NF-κB/cyclin D1 pathway and facilitate tumor growth. However, the effect of Daintain/AIF-1 on cell migration and cancer metastasis has never been reported. Herein, we used a mimic tumor microenvironment by incubating breast cancer cell lines, MDA-MB-231 and MCF-7 cells, with macrophage-conditioned medium with or without purified daintain/AIF-1 polypeptide to evaluate cell migration. Results indicated that daintain/AIF-1 enhanced the migration of MDA-MB-231 and MCF-7 cells in the manner of TNF-α up-regulation. Further study found that daintain/AIF-1 activates p38 MAPK signaling pathway contributing to up-regulation of TNF-α in MDA-MB-231 and MCF-7 cells. Therefore, this novel daintain/AIF-1-p38-TNF-α pathway and insight into daintain/AIF-1 might have potential benefits in the control of tumor metastasis during cancer therapy.